Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins. The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research. A subset of these glycoproteins has clustered sites of O-glycosylation with serine- and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation in cancer have made them potential targets as biomarkers for early detection of the disease. Immunoglobulin A1 (IgA1) contains both O- and N- glycans. Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes, as evidenced by formation of specific antibodies. Locating and characterizing the entire range of O-glycan attachment sites within this class of glycoproteins is analytically challenging due to the clustered serine, threonine, and often proline residues. We have recently developed protocols for the assessment of clustered IgA1 O-glycan macroheterogeneity (range and distribution of O-glycans attached within a 30-amino acid region) and microheterogeneity (range and distribution of O-glycan chains at each amino acid site within the same region) by use of high-resolution mass spectrometry and electron capture (or transfer) dissociation tandem mass spectrometry. Our recent progress with this challenge has led to the realization that a series of analytical tools for the analysis of clustered O-glycans in clinical samples needs to be standardized if proteins with clustered sites of O-glycosylation are to become reliable biomarkers. We propose the following specific aims to provide standardized guidelines for this class of post-translationally modified proteins in clinical samples: 1) Define the primary structure of clustered sites of O-glycosylation in IgA1 proteins from a series of clinical samples centered around patients with IgAN;2) Define the range of clustered O-glycan structures that are recognized by five different lectins;and 3) Develop strategies for the quantitative assessment of individual protein and peptide clustered O-glycoforms. PUBLIC HEALTH RELEVANCE: This proposal seeks to develop analytical tools for the analysis of clustered O-glycans in clinical samples that have become targets for their potential as biomarkers for cancer and other diseases. We will use high- resolution mass spectrometry and validated glycan-specific lectins to establish standards for the analysis of proteins with clustered sites of O-glycosylation. The goals are to identify and characterize the complete range of O-glycoprotein forms (for a single protein) in clinical samples, clearly defining lectin O-glycan recognition structures, and quantitatively analyze individual O-glycopeptides in clinical samples.